Bacteriochlorophyll-a derivatives useful in photodynamic therapy

ABSTRACT

Compounds of fomula (1) or formula (2): ##STR1## wherein M is a non-paramagnetic metal selected from Mg +2 , and Zn +2 , or represents 2 H +  each H +  bonded to one of the N atoms connected by the solid lines; 
     R 1  is a saturated or unsaturated hydrocarbyl residue of 8-25C; 
     each R 2  is independently selected from the group consisting of vinyl, ethyl, acetyl and 1-hydroxyethyl, and 
     X is COOR 3 , wherein R 3  is alkyl (1-4C); 
     are useful in photodynamic therapy and diagnosis. 
     These compounds photosensitize target biological substrates to irradiation, and treating said substrates with these sensitizers followed by irradiation leads to the impairment or destruction of the biological substrate. When administered systemically, these compounds accumulate in the undesired target biological substrate. The compounds can also be utilized in vitro, for example to destroy infectious cells or viruses in blood intended for transfusion.

TECHNICAL FIELD

The invention relates to the field of photodynamic therapy and relatedtreatment of in vitro samples using light-absorbing resonant ringsystems and irradiation. More specifically, the invention is directed tomethods of in vivo photodynamic therapy and diagnosis and in vitrosterilization using bacteriochlorophyll-a and its related compounds.

BACKGROUND ART

Photodynamic therapy using porphyrins and related compounds has, by now,a fairly long history. Early work, in the 1940s, demonstrated thatporphyrin could be caused to fluoresce in irradiated tumor tissue. Theporphyrins appeared to accumulate in these tissues, and were capable ofabsorbing light in situ, providing a means to detect the tumor by thelocation of the fluorescence. A widely used preparation in the earlystages of photodynamic treatment both for detection and for therapy wasa crude derivative of hematoporphyrin, also called hematoporphyrinderivative, HpD, or Lipson derivative prepared as described by Lipsonand coworkers in J Natl Cancer Inst (1961) 26:1-8. Considerable work hasbeen done using this preparation, and Dougherty and coworkers reportedthe use of this derivative in treatment of malignancy (Cancer Res (1978)38:2628-2635; J Natl Cancer Inst (1979) 62:231-237).

Dougherty and coworkers prepared a more effective form of thehematoporphyrin derivative which comprises a portion of HpD having anaggregate weight >10 kd. This form of the drug useful in photodynamictherapy is the subject of U.S. Pat. No. 4,649,151, is commerciallyavailable, and is in clinical trials.

The general principles of the use of light-absorbing compounds,especially those related to porphyrins, has been well established as atreatment for tumors when administered systemically. The differentialability of these preparations to destroy tumor, as opposed to normaltissue, is due to the homing effect of these preparations to theobjectionable cells. (See, for example, Dougherty, T. J., et al.,"Cancer: Principles and Practice of Oncology" (1982), V. T. de Vita,Jr., et al., eds., pp. 1836-1844.) Efforts have been made to improve thehoming ability by conjugating hematoporphyrin derivative to antibodies.(See, for example, Mew, D., et al., J Immunol (1983) 130:1473-1477.) Themechanism of these drugs in killing cells seems to involve the formationof singlet oxygen upon irradiation (Weishaupt, K. R., et al., CancerResearch (1976) pp. 2326-2329).

The use of hematoporphyrin derivative or its active components in thetreatment of skin diseases using topical administration has also beendescribed in U.S. Pat. No. 4,753,958. In addition, the drugs have beenused to sterilize biological samples containing infectious organismssuch as bacteria and virus (Matthews, J. L., et al., Transfusion (1988)28:81-83). Various other photosensitizing compounds have also been usedfor this purpose, as set forth, for example, in U.S. Pat. No. 4,727,027.

In general, the methods to use radiation sensitizers of a variety ofstructures to selectively impair the functioning of biologicalsubstrates both in vivo and in vitro are understood in the art. Thecompounds useful in these procedures must have a differential affinityfor the target biological substrate to be impaired or destroyed and mustbe capable of absorbing light so that the irradiated drug becomesactivated in a manner so as to have a deleterious effect on the adjacentcompositions and materials.

Because it is always desirable to optimize the performance oftherapeutics and diagnostics, variations on the porphyrin drugstraditionally used in treatment and diagnosis have been sought. A numberof general classes of photosensitizers have been suggested includingphthalocyanines, psoralen-related compounds, and multicyclic compoundswith resonant systems in general. Most similar to the compoundsdisclosed herein are various pheophorbide derivatives whose use inphotodynamic therapy has been described in EPO Application 220686 toNihon Metaphysics Company; ethylene diamine derivatives of pheophorbidefor this purpose described in Japanese Application J85/000981 to TamaSeikayaku, K. K., and Japanese Application J88/004805 which is directedto 10-hydroxy pheophorbide-a. In addition, pheophorbide derivatized to along chain hydrocarbyl group has been disclosed as useful inphotodynamic therapy in U.S. Ser. No. 221,804, filed Jul. 20, 1988,assigned to the same assignee and incorporated herein by reference. Inaddition, Beems, E. M., et al., in Photochemistry and Photobiology(1987) 46:639-643 discloses the use as photosensitizers of twoderivatives of bacteriochlorophyll-a --bacteriochloro-phyllin-a in whichthe exocyclic ring is opened and lacks the phytyl alcohol derivatized inbacteriochlorophyll-a) and bacteriochlorin-a (which is similar tobacteriochlorophyllin but lacks the Mg ion. These authors direct theirattention to these derivatives as being advantageous on the grounds ofenhanced water solubility as compared to bacteriochloro-phyll-a.

The problem remains to find suitable photosensitizers useful inphotodynamic therapy and diagnosis which are optimal for particulartargets and particular contexts. It is unlikely whether a singlecompound or small group of compounds, while generally applicable, wouldbe of maximum benefit in every instance. Thus, the invention provides anadditional group of photosensitizing compounds which becomes part of therepertoire of candidates for use in specific therapeutic and diagnosticsituations.

DISCLOSURE OF THE INVENTION

The invention provides alternative methods of photodynamic therapy anddiagnosis using a group of compounds related to thetetrahydroporphyrins, such as bacteriochlorophyll-a or -b or thecorresponding bacteriochlorins. These compounds are of formula (1) orformula (2): ##STR2## whereinM is a non-paramagnetic metal selected fromMg⁺², Sn⁺², and Zn⁺², or represents 2 H⁺, each H⁺ bonded to one of the Natoms connected by the solid lines;

R¹ is a saturated or unsaturated hydrocarbyl residue of 8-25C;

each R² is independently selected from the group consisting of vinyl,ethyl, acetyl and 1-hydroxyethyl, and

X is COOR³, wherein R³ is alkyl (1-4C).

Thus, in one aspect, the invention is directed to a method to effect theimpairment or destruction of a target biological substrate which methodcomprises treating the target substrate with an amount of the compoundof formula 1 effective to photosensitize said substrate followed byirradiating said target substrate with radiation in a wavelength bandabsorbed by the compound of formula 1 for a time effective to impair ordestroy the substrate.

In other aspects, the invention is directed to pharmaceuticalcompositions useful in the foregoing method, and to diagnostic kitswhich include the compound of formula 1 .

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-1 and 1-2 are tables showing the results of treatment withbacteriochlorophyll-a at various total radiation energies, wavelengthsand time intervals between injection of the sensitizer and lighttreatment.

FIG. 2 shows the action spectrum constructed from the table of FIG. 1.

FIG. 3 shows the tumor response as compared to foot sensitization tobacteriochlorophyll-a as a function of time.

MODES OF CARRYING OUT THE INVENTION

Bacteriochlorophyll-a (bchla) is a tetrahydroporphyrin found in certainphotosynthetic bacteria, for example, Rhodopseudomonas virdis. Bchla hasthe formula: ##STR3##

Bchla is essentially identical to the chlorophyll-a of higher plantsexcept that ring B is in the dihydro form and the vinyl group in ring Ais converted to an acetyl group. The wavelength absorption maximum ofbchla is about 780 nm and the extinction coefficient in this region isquite high (E₇₈₀ =75,000). This long wavelength absorption isadvantageous because light penetrates tissues 2-3 times more effectivelyat a wavelength near 800 nm versus lower wavelengths, e.g., 630 nm.

Bchla is readily obtained by extraction from bacterial sources, and isavailable commercially from Porphyrin Products, Logan, Utah. Althoughthe material is readily oxidized, especially in the presence of light,and the magnesium ion is readily removed in the presence of dilute acid,bchla is sufficiently stable in vivo to be an effective phototherapeuticagent.

In bacteriochlorophyll-b, which can also readily be obtained frombacterial sources, R² in the B ring is vinyl rather than ethyl. Theother embodiments of R² can easily be prepared starting withbacteriochlorophyll-b by standard hydration of the vinyl group to obtainthe 1-hydroxyethyl substituent, and mild oxidation to obtain thecorresponding acetyl substituent. Similarly, the R² substituent in ringA can be reduced to the 1-hydroxyethyl and/or dehydrated to vinyl and/orreduced to ethyl.

Conversion of the compounds of formula 1 to the compounds of formula 2can readily be effected by opening of the cyclopentanone ring usingknown reagents, such as alkaline solution in the presence of oxygen asdescribed in "Porphyrins and Metalloporphyrins", Smith, K., ed. (1975)Elsevier Press, pp. 52-53. Although the phytyl group is removed in thisreaction, reesterification to the desired R¹ can be effected by standardmethods.

In general, alternate embodiments of R¹ or R³ in either formula 1 orformula 2 can be obtained by transesterification or by hydrolysis andreesterification. In some instances, this esterification should beconducted on the compounds when they are in the form of thecorresponding porphyrin or dihydroporhryrins obtained by oxidation, forexample, using osmium tetroxide and then re-reducing to the tetrahydroform. In all of the conversions set forth above, it may be necessary toconduct the reactions in a certain order, to restore or remove the metalsubstituent and/or to utilize protective reactions and groups as isunderstood by practitioners in the art.

The compounds of formulas 1 and 2 are used for photodynamic therapy anddiagnosis with respect to target biological substrates. By "targetbiological substrate" is meant any cells, viruses or tissues which areundesirable in the environment to which therapy or other correctiveaction, such as sterilization, is employed, or the location of which isdesired to be known in an environment to which diagnosis is applied. Forexample, in a manner analogous to the use of the active fraction ofhematoporphyrin derivative (Hpd), as described in U.S. Pat. No.4,649,151, incorporated herein by reference, neoplastic tissue iseffectively treated in vivo by virtue of the ability of the drug toaccumulate preferentially in such tissue, and by virtue of thephotosensitizing nature of the drug. In this instance, the targetbiological substrate is the neoplastic tissue. As described in thispatent, the drug is injected into the subject, and permitted to clearnormal tissue. Then the neoplastic tissue is exposed to radiation at awavelength appropriate to its absorption spectrum. The patent furtherdescribes the synergistic effect of heat supplied, if desired, byinfrared irradiation. In addition, the location of the tumor can beascertained by the fluorescence of the drug.

In another application, Matthews, J. L., et al., Transfusion (1988)28:81-83, describe the use of the photosensitizing compounds HpD and theactive fraction thereof, designated DHE, in eradicating pathogens fromfluids in vitro. This article describes techniques for treating blood orother biological fluids to eliminate pathogens such as protozoa, virus,bacteria, fungi, and so forth. Similarly, U.S. Pat. No. 4,727,027describes the use of furocoumarin in conjunction with irradiation by UVlight for decontamination of blood products. In these instances, thetarget substrates are pathogens which may include a variety of"organisms" such as viruses and protozoa, as well as bacteria and fungi.

In U.S. Pat. No. 4,753,958, topical treatment of skin diseases usingphotosensitizing drugs is described. In this instance, the targetbiological substrate is the infectious virus or cell carrying thedisease. This too, may be a virus, bacterium, or other microorganism,including fungal infections.

For use in the method of the invention, the compounds of formula 1 and 2are formulated using conventional excipients appropriate for theintended use. For systemic administration, in general, buffered aqueouscompositions are employed, with sufficient nontoxic detergent tosolubilize the active compound. As the compounds of formulas 1 and 2 aregenerally not very soluble in water, a solubilizing amount of suchdetergent is employed. Suitable nontoxic detergents include Tween-80,various bile salts, such as sodium glycholate, various bile salt analogssuch as the fusidates. Alternate compositions utilize liposome carriers.The solution is buffered at neutral pH using conventional buffers suchas Hank's solution, Ringer's solution, or phosphate buffer. Othercomponents which do not interfere with the activity of the drug may alsobe included, such as stabilizing amounts of proteins, for example, serumalbumin.

Systemic formulations can be administered by injection, such asintravenous, intraperitoneal, intramuscular, or subcutaneous injection,or can be administered by transmembrane or transdermal techniques.Formulations appropriate for transdermal or transmembrane administrationinclude sprays and suppositories containing penetrants, which can oftenbe the detergents described above.

For topical local administration, the formulation may also contain apenetrant and is in the form of an ointment, salve, liniment, cream, oroil. Suitable formulations for both systemic and localized topicaladministration are found in Remington's Pharmaceutical Sciences, latestedition, Mack Publishing Co., Easton, Pa.

For use ex vivo to treat, for example, blood or plasma for transfusionor preparations of blood products such as Factor VIII, no specialformulation is necessary, but the compounds of formula 1 and 2 aredissolved in a suitable compatible solvent and mixed into the biologicalfluid at a suitable concentration, typically of the order of 1-100 ug/mlprior to irradiation.

For photodynamic therapeutic and diagnostic applications, suitabledosage ranges will vary with the mode of application and the choice ofthe compound, as well as the nature of the condition being treated ordiagnosed. However, in general, suitable dosages are of the order of0.1-50 mg/kg body weight, preferably 1-3 mg/kg. For topicaladministration, typically amounts on the order of 50-100 mg total areemployed.

The general procedures for photodynamic therapy and diagnosis in vivoare analogous to those described in U.S. Pat. No. 4,649,141; those forex vivo treatment are analogous to those described by Matthews, J. L.,et al., Transfusion (supra); topical methods are analogous to thosedescribed in U.S. Pat. No. 4,753,958; all are incorporated herein byreference.

Briefly, for systemic administration/ a suitable time period afteradministration, typically from several hours to two days is allowed toelapse in order to permit concentration of the drug of formula 1 or 2 inthe target biological substrate. In general, this substrate will be atumor, and the localization of the compound of formula 1 or 2 can bemonitored by measuring the fluorescence or absorption of the targettissue as compared to background. After localization has beenaccomplished, the target biological substrate is irradiated with asuitable band of irradiation, in the case of the compounds of formula 1,in the range of 750-800 nm at a rate of 5 mW/cm² -0.75 W/cm², and atotal energy of 100-1000 J/cm².

For topical treatment, localization is immediate, and the correspondingradiation can be provided immediately. For treatment of biologicalfluids ex vivo, again, no localization interval is required, andradiation is applied on the order of 1-10 J/cm². Because penetration oftissue is not required, lower total energy can be employed.

The following example, directed to bchla, is intended to illustrate butnot to limit the invention. The remaining compounds of formulas 1 and 2have similar absorption spectra as they contain the sametetrahydroporphyrin resonance system, and have similar solubilities.

EXAMPLE 1 Formulation of bchla

Bacteriochlorophyll-a, obtained at >90% purity from Porphyrin Products(Logan, Utah) was dissolved at a concentration of 5 mg/ml in Tween-80(Sigma) by stirring for several hours or overnight. The resultingsolution was mixed with 9 volumes of Hank's buffer solution withagitation until all of the detergent solution was dissolved. Anyremaining particulate matter is removed by filtration and theconcentration of the final solution is determined spectrophotometricallyusing a 1:100 dilution in distilled water (OD₇₈₀ =87.3 for 1 mg/ml ofconcentrate). In general, if the initial solution of bchla is conductedcarefully, the resulting formulation has a concentration of bchla of 0.5mg/ml.

EXAMPLE 2 Effect of bchla on Tumors

DBA2/HaD mice were transplanted with SMT-F tumors. When the subcutaneoustumors reached 4.5-5.5 mm in diameter, the mice, in groups of five, wereinjected intravenously with the bchla solution of Example 1 in doses of5-30 mg/kg. At a time 1 hour-5 days later, the tumor, previously shavedand depiliated, plus a margin of 2-3 mm was exposed to radiation of awavelength in the range 630-800 nm using a Spectraphysics argon dyelaser with Exciton LDS751 dye, tunable over the 700-800 nm range or adiode laser--e. g., Spectra Diode emitting in the 750-850 nm range or aXenon arc lamp filtered with an interference filter to pass 90% of the700 nm light ±60 nm at dose rates of 75-150 mW/cm². When the Xenonsystem was used, mild hyperthermia resulted (42° C. at 160 mW/cm²). Itis not known whether this temperature rise acts synergistically withbchla as has been shown with HpD and its active fraction.

Tumor response is shown in the table of FIG. 1 for the seventh day afterlight treatment which indicates regression, and at a time point at least30 days after light treatment, which would indicate cure, if there hadbeen no regrowth.

As shown in FIG. 1 good response to bchla was obtained, for example,after 2 hours at 5 mg/kg in the 670-790 nm range and after 24 hoursafter injection with 10 mg/kg and irradiated at 680-780 nm.

FIG. 2 shows the action spectrum along with the absorption spectra ofbchla, pheophytin (demetalated bchla, found in vivo) and for chlorophyll(oxidized bchla, theoretically found in vivo). The "X"s represent the 7day response when 270 J/cm² were used 2 hours after the administrationof 5 mg/kg; the squares represent the 7 day response when 270 J/cm² wereadministered 24 hours after administration of 10 mg/kg, and the circlesrepresent the 30 day (cure) response, all as a function of wavelength oflight used to treat the tumor.

EXAMPLE 3 Determination of Therapeutic Ratio

One of the undesirable side effects of photodynamic therapy usingcertain compounds is cutaneous photosensitivity unrelated to the targetbiological substrate. Accordingly, the effect of the treatment on thephotosensitivity of the foot of the treated mice was measured. Theresponse of the foot was measured as erythema and/or edema (or loss ofskin or further damage).

The results are shown in FIG. 3. The left ordinate shows the percentageof tumors which responded; the right ordinate is an arbitrary scale forthe foot response wherein 1.0 represents severe erythema and edema; 0.1represents little effect, and 0.5 represents a moderate reaction. Theresults show that for bchla, the sensitivity of the tumor and the skinof the foot declined concomitantly, while for the active component ofhematoporphyrin derivative designated DHE, the sensitivity persists formore than 10 days after injection. Thus, with DHE the tissue (foot)would be sensitive to light (for example, sunlight) for an extendedperiod of time (30 days in humans), whereas for bchla, sensitivity couldbe expected to persist for only a few days.

EXAMPLE 4 Metabolism of bchla

Uptake and clearance of bchla in tumor and liver were measured byextraction of the tumor or liver tissue with 1:1MeOH:CH₂ Cl₂, followedby HPLC analysis. The levels of bchla in tumor and liver after injectionof bchla are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        bchla Uptake in DBA/2 Ha Mice in SMT-F Tumor                                  Dose bchla                                                                              Time After    Tissue Level (ug/g)                                   (mg/kg)   Injection     Tumor    Liver                                        ______________________________________                                        10         2 h           6.14    44                                           10        24 h          --       49.4*                                        20         2 h          16       --                                           20        24 h          10-19.7* --                                           10        48 h          10.7*    --                                           ______________________________________                                         *Values at time intervals of 1 day or more are uncertain since preliminar     experiments indicate conversion to other components (see below).         

These results show that both tumor and liver have high levels of thecompound after 2 hours and that these levels are maintained for as longas 24 or 48 hours.

However, partial conversion to bacteriopheophytin occurs at 24 hours ormore in tumor and 2 hours in liver. Two hours after injection, the tumorcontains essentially only bchla with a small amount of material whereinthe phytyl group has hydrolyzed; at 48 hours the tumor contains mainlymaterial without phytyl and without Mg. At 24 hours the material intumor is demetallized but still contains phytyl.

EXAMPLE 5 Light Penetration

Comparison was made using bchla at 20 mg/kg with irradiation after 1hour at 270 J/cm² at 780 nm, and DHE at 5 mg/kg after 1 hour at 270J/cm² at 630 nm. Animals with tumors approximately 1 cm in depth wereused in the comparison. Histological sections were obtained the dayfollowing treatment, fixed and stained. A comparison using a total of 4animals showed a necrotic depth of 5-6 mm for DHE and approximately 9 mmfor bchla, consistent with deeper penetration of 780 nm light comparedto 630 nm light.

I claim:
 1. A method to effect the destruction or impairment ofundesired target biological substrates, which method comprises:a)treating said biological substrates with a compound of formula (1):##STR4## wherein M is a non-paramagnetic metal selected from Mg⁺², Sn⁺²,and Zn⁺², or represents 2 H⁺ each H bonded to one of the N atomsconnected by the solid lines;R¹ is a saturated or unsaturatedhydrocarbyl residue of 8-25C; each R² is independently selected from thegroup consisting of vinyl, ethyl , acetyl and 1-hydroxyethyl, and X isCOOR³, wherein R³ is alkyl (1-4C); said compound of formula (1) being inan amount effective to photosensitize said biological substrates to theresultant of irradiation absorbed by the compound of formula (1); and(b) irradiating the treated biological substrates with radiation havinga wavelength absorbed by the compound of formula (1).
 2. The method ofclaim 1 wherein the target biological substrates are contained in thebody of a mammalian subject and said treating is effected bysystematically administering the compound of formula (1), or apharmaceutical composition thereof, to said subject.
 3. The method ofclaim 1 wherein the target biological substrates are contained on theskin of a mammalian subject and said treating is effected by topicallyadministering the compound of formula (1), or a pharmaceuticalcomposition thereof, to said subject.
 4. The method of claim 1 whereinsaid biological substrates are contained in an in vitro biological fluidand said treating is conducted by adding the compound of formula (1) tothe biological fluid.
 5. The method of claim 4 wherein the biologicalfluid is blood or blood plasma.
 6. The method of claim 1 wherein thetarget biological substrate is selected from the group consisting oftumor cells, bacterial cells, fungi, protozoa and viruses.
 7. The methodof claim 1 wherein R¹ is a phytyl residue and M is Mg⁺².
 8. The methodof claim 1 wherein one R² is acetyl and the other R² is vinyl or ethyl.9. The method of claim 8 wherein the compound of formula (1) isbacteriochlorophyll-a or bacteriochlorophyll-b.
 10. The method of claim1 wherein the radiation is generated by a diode laser.